Changes in Natural Foxp3+Treg but Not Mucosally-Imprinted CD62LnegCD38+Foxp3+Treg in the Circulation of Celiac Disease Patients

BackgroundCeliac disease (CD) is an intestinal inflammation driven by gluten-reactive CD4+ T cells. Due to lack of selective markers it has not been determined whether defects in inducible regulatory T cell (Treg) differentiation are associated with CD. This is of importance as changes in numbers of induced Treg could be indicative of defects in mucosal tolerance development in CD. Recently, we have shown that, after encounter of retinoic acid during differentiation, circulating gut-imprinted T cells express CD62LnegCD38+. Using this new phenotype, we now determined whether alterations occur in the frequency of natural CD62L+Foxp3+ Treg or mucosally-imprinted CD62LnegCD38+Foxp3+ Treg in peripheral blood of CD patients. In particular, we compared pediatric CD, aiming to select for disease at onset, with adult CD.MethodsCell surface markers, intracellular Foxp3 and Helios were determined by flow cytometry. Foxp3 expression was also detected by immunohistochemistry in duodenal tissue of CD patients.ResultsIn children, the percentages of peripheral blood CD4+Foxp3+ Treg were comparable between CD patients and healthy age-matched controls. Differentiation between natural and mucosally-imprinted Treg on the basis of CD62L and CD38 did not uncover differences in Foxp3. In adult patients on gluten-free diet and in refractory CD increased percentages of circulating natural CD62L+Foxp3+ Treg, but normal mucosally-imprinted CD62LnegCD38+Foxp3+ Treg frequencies were observed.ConclusionsOur data exclude that significant numeric deficiency of mucosally-imprinted or natural Foxp3+ Treg explains exuberant effector responses in CD. Changes in natural Foxp3+ Treg occur in a subset of adult patients on a gluten-free diet and in refractory CD patients.

背景：乳糜泻（Celiac disease, CD）是一类由谷蛋白反应性CD4+ T细胞介导的肠道炎症性疾病。由于缺乏特异性筛选标志物，目前尚未明确诱导性调节性T细胞（inducible regulatory T cell, Treg）的分化缺陷是否与乳糜泻存在关联。这一问题具有重要研究价值，因为诱导性Treg的数量变化可反映乳糜泻患者黏膜耐受发育过程中的异常。近期本团队研究发现，在分化阶段接触维甲酸后，循环系统中的肠道归巢T细胞会表达CD62LnegCD38+表型。基于这一新的细胞表型，本研究旨在探究乳糜泻患者外周血中天然CD62L+Foxp3+ Treg或黏膜归巢CD62LnegCD38+Foxp3+ Treg的频率是否存在异常。本研究特别对比了儿童乳糜泻患者（纳入发病初期的病例）与成人乳糜泻患者的相关指标。

方法：采用流式细胞术（flow cytometry）检测细胞表面标志物、胞内Foxp3及Helios的表达水平；同时通过免疫组织化学法（immunohistochemistry）检测乳糜泻患者十二指肠组织中Foxp3的表达情况。

结果：儿童乳糜泻患者与年龄匹配的健康对照者的外周血CD4+Foxp3+ Treg占比无显著差异。基于CD62L与CD38对天然Treg与黏膜归巢Treg进行分型的分析未发现Foxp3表达存在异常。在接受无谷蛋白饮食治疗的成人患者以及难治性乳糜泻患者中，循环系统中天然CD62L+Foxp3+ Treg的占比升高，但黏膜归巢CD62LnegCD38+Foxp3+ Treg的频率维持正常水平。

结论：本研究数据排除了"黏膜归巢型或天然Foxp3+ Treg的显著数量缺失可解释乳糜泻中过度的效应性免疫应答"这一假说。天然Foxp3+ Treg的数量变化仅见于部分接受无谷蛋白饮食治疗的成人患者以及难治性乳糜泻患者。